¹Institute of Pathology, Charité Campus Mitte, Humboldt University Berlin, D-10117 Berlin, Germany

²Max-Delbrück-Center for Molecular Medicine, D-13092 Berlin, Germany

³Free University Medical Center, Department of Pathology, 1007 Amsterdam, The Netherlands

Correspondence to: Dr Hermann Lage, Institute of Pathology, Charité Campus Mitte, Humboldt University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: hermann.lage@charite.de

The phenomenon of multidrug resistance (MDR) in human cancers is one of the major causes of failure of chemotherapy. A recently identified new member of the superfamily of ATP-binding cassette transporters, breast cancer resistance protein (BCRP), was demonstrated to confer an atypical multidrug-resistant phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, a specific anti-BCRP hammerhead ribozyme was introduced into the human gastric carcinoma cell line, EPG85-257RNOV, exhibiting an atypical MDR phenotype. By this approach, the expression levels of the targeted BCRP-encoding mRNA and the BCRP transport protein were decreased to the low constitutive expression level that was observed in highly drug-sensitive parental gastric carcinoma cells. In addition, in the anti-BCRP ribozyme-treated cells, the cellular drug accumulation was dramatically increased to the level measured in drug-sensitive cells. These effects were accompanied by an extensive reversal of the drug-resistant phenotype of more than 80%. Because additional mechanisms contribute to the multimodal-mediated MDR phenotype exhibited by this gastric carcinoma cell line, the data suggest that the BCRP-mediated contingent to the drug resistance was overcome nearly completely. Moreover, the data indicate that ribozyme-based gene therapy may be clinically applicable in preventing and reversing BCRP-mediated atypical MDR. Cancer Gene Therapy (2002) 9, 579-586 doi:10.1038/sj.cgt.7700471

BCRP; ribozyme; atypical multidrug resistance; mitoxantrone; gastric carcinoma

Multidrug resistance (MDR) of cancer cells is one of the major causes for failure in chemotherapeutic treatment of human malignancies. Simultaneous resistance of tumor cells to various antineoplastic agents that are structurally and functionally unrelated characterizes this phenomenon. Enhanced expression of the ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp) (overview in ^{Ref. [1]}), causes the so-called classical MDR phenotype. Moreover, alternative forms of MDR have been described. These types of MDR are not attributed to P-gp overexpression, and are known as atypical MDR or non-P-gp-mediated MDR. Members of the superfamily of ABC transporters can act as energy-dependent xenobiotic efflux pumps. This transport activity results in decreased intracellular concentrations of antineoplastic agents. Various ABC transporters are involved or even responsible for MDR, e.g., transport molecules of the multidrug resistance protein (MRP) subfamily (overview in ^{Ref. [2]}), as well as the breast cancer resistance protein (BCRP, MXR, ABCG2), have been shown to be associated with atypical MDR phenotypes. BCRP (overview in ^{Ref. [3]}) is a 72-kDa ABC "half-transporter" consisting of 655 amino acid residues, which is thought to homo- or heterodimerize to form an active transport complex. The protein is overexpressed in a variety of atypical multidrug-resistant human cancer cell lines, which were established by in vitro exposure to mitoxantrone, topotecan, doxorubicin, or bisantrene.⁴ Transfection experiments demonstrated that the BCRP-encoding cDNA can confer an atypical MDR phenotype to formerly drug-sensitive cancer cells.⁵

Several pharmacologically active products, designated as MDR modulators or chemosensitizers, may circumvent the classical MDR phenotype (overview in ^{Ref. [6]}). One obstacle in applying MDR modulators, such as calcium channel inhibitors, arises from their commonly occurring intrinsic toxicity at doses necessary to be effective. Additionally, tumor cells can develop resistance against the applied chemosensitizers, a so-called tertiary resistance. In the case of reversing BCRP-mediated atypical MDR, there is a limited number of compounds available showing BCRP-inhibiting effects: the Aspergillus fumigatus secondary metabolite fumitremorgin C,⁷ its derivative demethoxy-fumitremorgin C,⁸ the so-called second-generation MDR modulator GF120918,⁹ the quinazoline-based HER family tyrosine kinase inhibitor CI1033,¹⁰ and experimental camptothecin analogues.^11,12

Consequently, it is necessary to develop alternative and efficient strategies to overcome atypical MDR. In recent years, antisense oligodeoxynucleotides and hammerhead ribozymes have been successfully applied in decreasing the expression level of genes involved in malignancy and therapy resistance, including drug resistance-associated genes.^{13,14,15,16,17} One considerable advantage of hammerhead ribozymes over antisense oligodeoxynucleotides is their intrinsic endoribonucleolytic cleavage activity. Hammerhead ribozymes can be designed to cleave a specific mRNA molecule at a defined position in trans, provided that the target molecule contains a NUX motif, in which "N" is any nucleotide (nt) and "X" is A, C, or U.¹⁸ Consequently, modulation of BCRP gene expression by the treatment with antisense oligonucleotides or hammerhead ribozymes may decrease BCRP-mediated drug efflux activity and reverse the tumor cell's drug resistance.

Antisense oligonucleotides directed against the BCRP-encoding mRNA have already been applied to modulate resistance against SN-38, the active metabolite of the camptothecin derivative irinotecan.¹⁹ However, the ribozyme technology should reveal a higher efficiency in reversing drug resistance.

To confirm this hypothesis, we previously constructed a hammerhead ribozyme, designated RzB1, directed against the BCRP-encoding mRNA.²⁰ The in vitro characterization of the anti-BCRP hammerhead ribozyme RzB1 revealed a high endoribonucleolytic activity in a cell-free system. In the present work, we report the design and insertion of this anti-BCRP hammerhead ribozyme into a eukaryotic expression vector and the implementation of this construct into the BCRP-overexpressing human gastric carcinoma cell line, EPG85-257RNOV (257RNOV).⁴

Materials and methods

Cell lines and drugs

The establishment and culture of the human gastric carcinoma cell line, EPG85-257P (257P), and its atypical MDR variant, EPG85-257RNOV (257RNOV), were described in detail previously.²¹ Transfected cell clones were grown under the same conditions and additionally in the presence of 400 g/mL G418 (GibcoBRL, Grand Island, NY). The following antineoplastic agents were used: mitoxantrone (Lederle, Wolfratshausen, Germany), etoposide and daunorubicin (Farmitalia Carlo Erba, Freiburg, Germany), and cisplatin (Bristol-Myers, München, Germany).

Construction of a ribozyme and control expression vector systems

Cell clones were obtained by stable transfection of the ribozyme RzB1 targeted to BCRP into the eukaryotic expression vector, pcDNA3.1 (Invitrogen, San Diego, CA). The ribozyme's catalytic activity was previously characterized in a cell-free system.²⁰ The RzB1-encoding sense and antisense oligodeoxynucleotides (RzB1 sense: 5'-CTG GAA CTG ATG AGT CCG TGA GGA CGA AAC ATC TGG AGA-3'; RzB1 antisense: 5'-CTC CAG ATG TTT CGT CCT CAC GGA CTC ATC AGT TCC AGA-3') were synthesized chemically with overhanging A-ends. The coding and the corresponding strands (each at 50 M) were hybridized for 10 minutes at 60°C, and cloned into the multiple cloning side of a prepared T-tailed pcDNA3.1 vector provided in the eukaryotic TOPO TA Cloning Kit (Invitrogen). Correct sequence and insertion of RzB1 into the pcDNA3.1 vector were confirmed by sequencing using an ABI-373 sequencer (Perkin Elmer, Foster City, CA). Controls (inverted RzB1/inv and mutated RzB1/mut) were constructed in the same manner.

Plasmid transfection and selection of stable transfected cell clones

Atypical multidrug-resistant gastric carcinoma cells, 257RNOV, were transfected with 1 g of expression vector DNA (RzB1, RzB1 in sense orientation related to the CMV promoter; RzB1/inv, RzB1 in antisense orientation related to the CMV promoter; RzB1/mut, RzB1 in sense orientation related to the CMV promoter, but containing two point mutations inhibiting the ribozyme's catalytic activity). Each transfection experiment was performed in 70-80% confluent 35-mm cell culture plates using 5 L of the transfection reagent SuperFect (Qiagen, Hilden, Germany). The transfected cell clones were selected for 3 weeks in G-418-containing (400 g/mL) cell culture medium. After this period, visible clones were picked, expanded in 24-well plates, and finally transferred to regular cell culture flasks. Entirely 22 anti-BCRP hammerhead ribozyme RzB1-expressing clones and six clones of each control variant expressing RzB1/inv orRzB1/mut were isolated and expanded. Each of these clones was analyzed separately. In each case, a representative clone was chosen and used for the detailed characterization of the cellular effects of the utilized ribozyme constructs.

Confirmation of ribozyme expression

Expression of the anti-BCRP ribozyme RzB1 in the G418-resistant clones (257RNOV-RzB1) and the control-transfected clones (257RNOV-RzB1/inv, 257RNOV-RzB1/mut) was controlled by RT-PCR using vector-specific oligodeoxynucleotide primers T7-fw, 5'-TAA TAC GAC TCA CTA TAG GG-3' and BGH-rev, 5'-TAG AAG GCA CAG TCG AGG-3', which yielded an expected 304-bp PCR product. As positive control for each cell line, aldolase-specific primers were used (Ald-fw, 5'-GGC AAG GGC ATC CTG GCT GCA CA-3' and Ald-rev, 5'-TAA CGG GCC AGA ACA TTG GCA T-3'), yielding an expected amplification product of 443 bp.

Detection of BCRP mRNA expression level

Levels of BCRP-encoding mRNA expression were determined by Northern blot analyses applying standard procedures as described previously.²² A 540-bp BCRP-specific hybridization probe was generated by PCR. The template was a BCRP-encoding cDNA fragment prepared from the atypical MDR cell line, 257RNOV, that was cloned into the pcDNA3.1 vector.³ Oligodeoxynucleotide primers used for amplification were M13-fw, 5'-CAG GAA ACA GCT ATG AC-3' and M13-rev, 5'-CAA AAG GGT CAG TGC TG-3'.

Immuno flow cytometry

BCRP expression was measured by immuno flow cytometry. Cells were prepared as described previously²³ and incubated with 1:10 diluted BCRP-specific mouse monoclonal antibodies (mAbs) BXP-21²⁴ and BXP-34²⁵ for 60 minutes at 4°C. A fluorescein-conjugated goat-antimouse mAb (Biosource International, Nivelles, Belgium) was utilized to detect the primary anti-BCRP mAb. Measurement of the fluorescence intensity of 10⁴ cells per clone was performed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and expressed as mean fluorescence per cell. Values were calculated as difference from the mean fluorescence per cell for the mAb of interest and a negative control mouse IgG₁ (Becton Dickinson).

Cytotoxicity assay for cell survival

Chemoresistance was tested using a proliferation assay based on sulforhodamine B (SRB), a protein-binding reagent.²⁶ In each experiment, 350 cells/well were seeded in 96-well plates in complete medium and allowed to attach overnight. Mitoxantrone or various drugs (etoposide, daunorubicin, cisplatin) were added in a dilution series in triplicate wells. After 5 days, when unselected wells were still subconfluent, incubation was terminated by replacing the medium with 10% trichloroacetic acid, followed by incubation at 4°C for 1 hour. Subsequently, the plates were washed five times in water and stained by adding 100 L of 0.4% SRB (Sigma, St. Louis, MO) in 1% acetic acid for 10 minutes at room temperature. Unbound dye was eliminated by washing the plates five times with 1% acetic acid. After air-drying and resolubilisation of the protein-bound dye in 10 mM Tris-HCl (pH 8.0), absorbance was read at 562.5 nm. To determine the IC₅₀ value, the absorbance difference of control cells without drug was set to be 100%. Linear regression was plotted for the linear region of the curve, and IC₅₀ values were calculated from multiple, at least three independent, experiments for each cell line.

Drug accumulation assays

Cellular mitoxantrone accumulation was determined according to the procedure originally reported by Allen et al,²⁷ with some modifications as described previously.²⁸ Briefly, 4´10⁵ cells were seeded in six-well plates 24hours prior to the experiment. Incubation with mitoxantrone (0, 1, 5, and 10 g/mL) was performed for 60 minutes at 37°C and 70-80% cellular confluence. For time kinetics, cells were exposed to 10 g/mL mitoxantrone for 0, 5, 10, and 60min. Cells were washed with ice-cold PBS, trypsinized, and resuspended in 4°C medium at a concentration of 4´10⁵ cells/mL. Intracellular fluorescence of mitoxantrone was determined by flow cytometry (Becton Dickinson). The cells were excited at 480 nm and emission was measured at 630 nm. A minimum of 10⁴ cells was analyzed for each sample. All assays were performed at least in three independent experiments, each time with triplicate wells.

Results

Expression of the anti-BCRP ribozyme RzB1 in atypical MDR cells

The human gastric carcinoma cell line, 257RNOV,²¹ exhibits an atypical multidrug-resistant phenotype, which is accompanied by a considerable overexpression of the BCRP-encoding mRNA.⁴ This cell line was transfected with a CMV promoter-driven eukaryotic expression vector carrying the anti-BCRP ribozyme RzB1 (Fig 1). Recent studies revealed a high catalytic activity of the ribozyme RzB1 in a cell-free system.²⁰ As controls, two further expression vectors were constructed in the same way, but containing the following modifications: one vector was designed with the anti-BCRP ribozyme RzB1-encoding sequence in antisense orientation to the CMV promoter; the other sense-oriented ribozyme insert contains two point mutations (Fig 1) that completely inhibit the ribozyme's cleavage activity (G₅U and A₁₄C).^18,29 Stable expression of the hammerhead ribozymes in the transfected clones was confirmed by RT-PCR (Fig 2A). Names of the cell lines were as follows: 257P, parental, nonresistant gastric carcinoma cell line EPG85-257P²¹; 257RNOV, atypical MDR variant EPG85-257RNOV; 257RNOV-RzB1, atypical MDR 257RNOV cells expressing the anti-BCRP ribozyme RzB1; 257RNOV-RzB1/mut, 257RNOV-derived control clone expressing a catalytic inactive mutated ribozyme; 257RNOV-RzB1/inv, 257RNOV-derived control clone expressing an inverted ribozyme construct.

Effect of anti-BCRP ribozyme RzB1 on the expression of the BCRP-encoding mRNA and the BCRP polypeptide

Northern blot experiments were performed to analyze the expression of the BCRP-encoding mRNA in the nonresistant parental cell line 257P, the atypical multidrug-resistant gastric carcinoma cell line 257RNOV, the anti-BCRP ribozyme RzB1-treated cells, and the control clones. As shown in Figure 2B, a high BCRP mRNA-specific expression level could be detected in drug-resistant 257RNOV cells and the control clone 257RNOV-RzB1/inv treated with an inverted ribozyme without any catalytic or antisenseproperties. In contrast, the nonresistant cell line 257P and the catalytically active anti-BCRP ribozyme-treated cell variant, 257RNOV-RzB1, displayed a considerably decreased expression level of the BCRP-encoding mRNA. However, the second control clone 257RNOV-RzB1/mut treated with an endoribonucleolytic inactive mutated anti-BCRP hammerhead ribozyme showed a slight decrease of the BCRP mRNA level compared to the original atypical multidrug-resistant cell line, 257RNOV. This effect might bethe result of putative antisense effects of the 5' and 3' armsof the catalytically inactive ribozyme sequence.

Immuno flow cytometry experiments using the BCRP-specific mAbs BXP-21²⁴ and BXP-34²⁵ demonstrated that the anti-BCRP ribozyme RzB1-transfected clone decreased the cellular BCRP protein content to approximately 24% (for BXP-21) or 23% (for BXP-34) of the origin level in the atypical multidrug-resistant cell line, 257RNOV (P=.0022) (Fig 3). Likewise, this BCRP expression value is even lower than in the parental, nonresistant human gastric carcinoma cell line, 257P. This observation indicates that the anti-BCRP hammerhead ribozyme RzB1, which already showed a high endoribonucleolytic cleavage activity in a cell-free system,²⁰ also cleaves its specific substrate mRNA molecule very efficiently in a cellular environment. In conformity with the data obtained by Northern blot analyzes, cell clones transfected with a mutated, catalytic inactive ribozyme, 257RNOV-RzB1/mut, also showed a decreased BCRP expression level when compared to the drug-resistant, nontransfected cell line, 257RNOV (46% of the original BCRP expression level of 257RNOV cells using BXP-21; 74% of the protein expression value using BXP-34). However, no significant modulation of the BCRP expression was detected in the cell clone, 257RNOV-RzB1/inv, transfected with an inverted variant of the anti-BCRP ribozyme, RzB1.

Sensitivity of anti-BCRP ribozyme RzB1-treated cells to anticancer drugs

As shown in Figure 4 and Table 1, the anti-BCRP ribozyme-treated cell clone, 257RNOV-RzB1, decreased the resistance level against mitoxantrone to approximately 18% of the mitoxantrone-specific IC₅₀ value assessed in the atypical multidrug-resistant cell line, 257RNOV. The drug resistance against etoposide, cisplatin, and daunorubicin was not modulated significantly.

Accumulation of mitoxantrone in anti-BCRP ribozyme RzB1-treated clones

Figure 5 demonstrates that the mitoxantrone accumulation was dramatically enhanced in the anti-BCRP hammerhead ribozyme-treated gastric carcinoma cell clone, 257RNOV-RzB1, when compared to the atypical MDR cell line, 257RNOV. Time-dependent as well as drug concentration-dependent experiments revealed a similar mitoxantrone accumulation level in the clone, 257RNOV-RzB1, compared to the nonresistant, parental carcinoma cell line, 257P. The inverted ribozyme-expressing control clone, 257-RzB1/inv, accumulated a similar amount of drug as the drug-resistant cell variant, 257RNOV. As expected, the cell variant, 257RNOV-RzB1/mut, treated with the endoribonucleolytic inactive but potential antisense effects causing anti-BCRP ribozyme showed a moderately elevated drug accumulation rate.

Discussion

The experimental endeavors to overcome the toxic effects of conventional MDR modulators resulted in the development of antisense and antigene strategies by applying oligodeoxynucleotides^16,30 and ribozymes^{13,14,15,20,31,32} that are directed against the transcripts of genes encoding MDR-associated factors. The data presented in this study demonstrate the modulation of the expression of the atypical MDR-associated ABC transporter BCRP in human cancer cells by a hammerhead ribozyme-based gene therapeutic approach. It could be shown that the anti-BCRP hammerhead ribozyme RzB1, featured by a high endoribonucleolytic activity in a cell-free system,²⁰ also worked efficiently in a human tumor cell system. The BCRP-mediated drug-resistant phenotype could be extensively reversed to more than 80% by decreasing the amount of the BCRP-encoding mRNA. This resulted in a decrease of the expression of the derived ABC transporter molecule as well as an increase in the cellular drug accumulation rate. In addition, the extent of the reversal of drug resistance in the cell line, 257RNOV, suggests that BCRP is one of the major mechanisms contributing to the atypical MDR phenotype, especially the cross-resistance against mitoxantrone, in this gastric carcinoma cell line.

Gene expression analyses also revealed a slightly decreased BCRP mRNA expression level as well as protein expression level in the control clone, treated with a ribonucleolytic inactive ribozyme (RzB1/mut). Antisense effects of the 5' and 3' flanking sequences of the catalytic inactive control ribozyme could explain this phenomenon. In contrast to the behavior of the inactive ribozyme RzB1/mut-treated clone, the inverted ribozyme RzB1/inv-treated clone did not show any alteration in BCRP mRNA or protein expression when compared to the drug-resistant cell line, 257RNOV, indicating that no unspecific side effects may occur due to the transfection procedure. Such unspecific clonal effects represent a general problem by performing transfection experiments. The fact that approximately one third of the anti-BCRP-transfected clones and none of the controls reduced their vulnerability to mitoxantrone treatment but not to exposure to daunorubicin, etoposide, or cisplatin supports the idea that the new phenotype exhibited by the ribozyme-transfected clones was indeed caused by the ribozyme and not by such clonal effects.

The BCRP mRNA and protein expression level of anti-BCRP ribozyme-expressing cells was similar to that observed in the nonresistant cell line, 257P, but did not result in the same drug-resistant phenotype. However, this observation is not astonishing because it was reported previously that alternative mechanisms, such as compartmentalization of the drug,²¹ alterations in DNA topoisomerase II expression,³³ or enhanced activity of the ABC transporter "transporter associated with antigen presentation" (TAP),²⁸ contribute to the drug-resistant phenotype in this cell line. Therefore, a complex interaction of different molecular mechanisms mediates the drug-resistant phenotype exhibited by the human gastric carcinoma cell line, 257RNOV. Because it is obvious that the atypical MDR phenotype exhibited by the gastric carcinoma cell line, EPG85-257RNOV, is multimodal-mediated, the data suggest that the BCRP-mediated contingent to the drug-resistant phenotype was reversed nearly completely by the cellular introduction of the anti-BCRP hammerhead ribozyme RzB1. As expected, a weak decrease of mitoxantrone resistance could be observed in the control clone, 257RNOV-RzB1/mut, in which antisense effects of the catalytic inactive ribozyme could have caused a decrease in BCRP expression. The weak cross-resistance against the BCRP substrates etoposide and daunorubicin was not influenced in any of the ribozyme-treated cells. The higher impact of DNA topoisomerase II in etoposide- and daunorubicin resistance compared to mitoxantrone could explain this effect. In other words, to influence etoposide- and daunorubicin resistance, the decrease in BCRP expression was insufficient in the analyzed cells. As expected, the cisplatin-resistant phenotype remained unaltered in all transfected cell clones because this resistance is commonly caused by alternative resistance mechanisms, e.g., by a modulation of the activity of the DNA mismatch repair system (overview in ^{Ref. [34]}).

The reversal of drug resistance in the anti-BCRP ribozyme RzB1-treated clone, 257RNOV-RzB1, was reflected by a dramatic time- and concentration-dependent increase of mitoxantrone accumulation. In conformity with the alterations in the drug-resistant phenotypes of ribozyme-transfected cell clones, no dramatic changes in cellular mitoxantrone accumulation could be observed in the clone, 257RNOV-RzB1/inv, whereas mitoxantrone fluorescence increased in the catalytic inactive ribozyme-expressing clone, 257RNOV-RzB1/mut, that nevertheless could exhibit antisense effects.

Recently, BCRP was identified and characterized as a novel stem cell transporter.³⁵ In a murine system, it could be demonstrated that BCRP expression was highly conserved in primitive stem cells from a variety of sources including hematopoietic and embryonic stem cells. The level of BCRP protein expression directly correlated with the stem cell-characterizing side population phenotype. This observation has to be taken into consideration when BCRP-inhibiting agents, conventional pharmacological compounds, or antisense/ribozyme constructs are applied in the clinical situation. Although the normal physiologic role of BCRP in stem cells has not been clarified, the available data suggest a potential functional role in hematopoiesis. On that score, there is the potential danger of a pancytopenia following combined chemotherapy using a BCRP-modulating agent.

In conclusion, the data demonstrated the proof of principle that endoribonucleolytic active hammerhead ribozymes can be designed to cleave the BCRP-encoding mRNA in targeted tumor cells. The resulting decrease of the cellular BCRP content led to an enhanced drug accumulation that reversed the drug-resistant phenotype extensively. Hence, the utilized anti-BCRP hammerhead ribozyme RzB1 appears to be an effective laboratory tool for the investigation of the relationship between BCRP expression and the atypical MDR phenomenon. In addition, ribozyme technology has implications for the prevention and reversal of atypical MDR in animals and man by gene therapeutic approaches.

Acknowledgements

This work was supported in part by grants of the "NOVARTIS Stiftung für therapeutische Forschung", the "Deutsche Forschungsgemeinschaft" (LA 1039/2-1) and the "Deutsche Krebshilfe" (10-1313-La3). We are grateful to RJ Scheper (Free University Amsterdam, the Netherlands) for providing the BCRP-specific mAbs BXP-21 and BXP-34.

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Figure 1 Schematic representation of the hammerhead ribozyme, RzB1, directed against the BCRP-encoding mRNA. The cleavage site in the target mRNA is indicated by an arrow; the GUC recognition triplet is underlined. The numbering of the hammerhead ribozyme is according to the system suggested by Hertel et al.³⁶ For construction of a noncatalytic active control ribozyme, two point mutations were introduced in the sequence, G₅U and A₁₄C. The BCRP-encoding sequence and nt positions were derived from the BCRP cDNA sequence (GenBank accession no. AF098951).⁵ The nt position +1 is assigned to the A residue of the ATG translation start codon.

Figure 2 Effect of the BCRP-specific hammerhead ribozyme RzB1 on BCRP-encoding mRNA expression level. A: RT-PCR confirmation of ribozyme expression in the atypical MDR gastric carcinoma cell line, 257RNOV; 257RNOV-RzB1/inv, 257RNOV transfected with the ribozyme RzB1 in antisense orientation related to the CMV promoter; 257RNOV-RzB1/mut, 257RNOV transfected with a catalytic inactive ribozyme; the anti-BCRP hammerhead ribozyme RzB1-expressing clone 257RNOV-RzB1; and the parental, nonresistant cell line, 257P. As a control, RT-PCR directed against the mRNA encoding the housekeeping enzyme aldolase was performed. B: A representative Northern blot analysis demonstrating the decrease of BCRP mRNA expression level in anti-BCRP ribozyme RzB1-transfected cells. For control of the equivalence in sample loading, the blot was stripped and rehybridized using a cDNA encoding the housekeeping enzyme, phosphoglycerate kinase (PGK).

Figure 3 BCRP expression as measured by immuno flow cytometry using two different mAbs BXP-21 and BXP-34 directed against BCRP in the atypical MDR gastric carcinoma cell line, 257RNOV; 257RNOV-RzB1/inv, 257RNOV transfected with the ribozyme RzB1 in antisense orientation related to the CMV promoter; 257RNOV-RzB1/mut, 257RNOV transfected with a catalytic inactive ribozyme; 257RNOV-RzB1, the anti-BCRP hammerhead ribozyme RzB1-expressing clone; and the parental, nonresistant cell line, 257P. BCRP polypeptide expression values are means of at least three independent experiments using both antibodies each; error bars show SD.

Figure 4 Reversal of the drug-resistant phenotype by utilization of the anti-BCRP hammerhead ribozyme, RzB1. Cytotoxicity of (A) mitoxantrone, (B) daunorubicin, (C) etoposide, and (D) cisplatin was determined by a cell proliferation assay. 257P, nonresistant, parental cell line; 257RNOV, atypical MDR variant; 257RNOV-RzB1, atypical MDR cells transfected with an active hammerhead ribozyme directed against BCRP; 257RNOV-RzB1/mut, atypical MDR cells transfected with a catalytic inactivated ribozyme; 257RNOV-RzB1/inv, atypical MDR cells transfected with anti-BCRP ribozyme RzB1 in antisense orientation to the CMV promoter. Error bars represent SD of at least three triplicate determinations of cell proliferation.

Figure 5 Accumulation of mitoxantrone as measured by flow cytometry in the nonresistant, parental cell line 257P and its atypical MDR derivative 257RNOV as well as anti-BCRP ribozyme RzB1 transfectants, 257RNOV-RzB1, and control clones (257RNOV-RzB1/mut, 257RNOV-RzB1/inv) derived from the atypical MDR cell line. A: Drug accumulation as a function of time by incubation with 10 g/mL mitoxantrone. B: Drug accumulation as a function of increasing concentrations of mitoxantrone after 60-minute drug incubation. Displayed values represent the mean; the vertical error bars represent SD of at least three replicate experiments.

Table 1 Cross-resistance of anti-BCRP ribozyme-treated cells